Bottom Line:
This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion.Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B.Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

ABSTRACTSeveral kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

fig1: Plk1 binds to vimentin phosphorylated by Cdk1. (A) Site-specific phosphorylation of vimentin during metaphase/anaphase in U251 cells. (B) Metaphase or postmitotic T24 cells expressing vimentin WT or vimentin mutated at Cdk1 (mutant I: Ser41 and Ser55) or Rho-kinase + Aurora-B (mutant II: Ser6, Ser24, Ser38, Ser46, Ser64, Ser65, Ser71, Ser72, and Ser86) phosphorylation sites (Yasui et al., 2001). Green color represents the staining with anti-vimentin (A and B) or the site- and phosphorylation state–specific antibody for Ser55, Ser71, or Ser72 on vimentin (A). DNA was also stained with propidium iodide (red). (C) Specific interaction of Plk1 with vimentin phosphorylated by Cdk1. Vimentin was incubated with (Cdk1) or without (Control) Cdk1. Each sample was then subjected to immunoblotting with 4A4 (P-ser55) or Far Western blotting using GST-PBD. (D) Site specificity of plk1 determined by competition assay. After Far Western blotting (C and D), transferred membrane was stained with Coomassie brilliant blue (CBB). (E) GST pull-down assay using GST-PBD. Each sample was prepared as described in Materials and methods, and then subjected to immunoblotting with 4A4 (P-Ser55) and anti-vimentin antibody.

fig1: Plk1 binds to vimentin phosphorylated by Cdk1. (A) Site-specific phosphorylation of vimentin during metaphase/anaphase in U251 cells. (B) Metaphase or postmitotic T24 cells expressing vimentin WT or vimentin mutated at Cdk1 (mutant I: Ser41 and Ser55) or Rho-kinase + Aurora-B (mutant II: Ser6, Ser24, Ser38, Ser46, Ser64, Ser65, Ser71, Ser72, and Ser86) phosphorylation sites (Yasui et al., 2001). Green color represents the staining with anti-vimentin (A and B) or the site- and phosphorylation state–specific antibody for Ser55, Ser71, or Ser72 on vimentin (A). DNA was also stained with propidium iodide (red). (C) Specific interaction of Plk1 with vimentin phosphorylated by Cdk1. Vimentin was incubated with (Cdk1) or without (Control) Cdk1. Each sample was then subjected to immunoblotting with 4A4 (P-ser55) or Far Western blotting using GST-PBD. (D) Site specificity of plk1 determined by competition assay. After Far Western blotting (C and D), transferred membrane was stained with Coomassie brilliant blue (CBB). (E) GST pull-down assay using GST-PBD. Each sample was prepared as described in Materials and methods, and then subjected to immunoblotting with 4A4 (P-Ser55) and anti-vimentin antibody.

Bottom Line:
This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion.Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B.Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

ABSTRACTSeveral kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.